Horseradish Peroxidase: Dynamics and Nonaqueous Activity

描述(由申请人提供)：蛋白质组学的预期突破对人类健康的改善将取决于对蛋白质的展开/折叠和动力学以及非水溶剂对酶动力学的影响的更全面的了解。这样的理解将从模型酶辣根过氧化物酶(HRP)的研究中寻求。我们将遵循在展开过程中二级和三级结构变化的时间顺序，关联非水溶剂引起的二级和三级结构和酶活性的变化，并研究底物类似物在蛋白质内扩散的动力学。实验方法将包括荧光和磷光、圆二向色性(CD)和UV/可见光吸收。最终，停流设备将被用来测量更快的分子事件。具体目的是检验五个假说。1)通过监测色氨酸荧光、近/UV-CD、远/UV-CD和Soret-CD吸光度的相对时程，可以确定HRP在去折叠过程中二级结构和三级结构变化的时间顺序。2)非水溶液中HRP的构象变化可以测量到，并与酶活性的显著降低有关。3)三氟乙醇(TFE)改变了HRP的血红素可及性，改变了其酶活性。4)TFe可与其他底物一起改变HRP的酶活性。5)HRP底物和底物类似物的结构可以与它们通过HRP底物通道扩散的激活能相关联。

DESCRIPTION (provided by applicant): Improvements to human health from the anticipated breakthroughs in proteomics will depend on a fuller understanding of unfolding/folding and dynamics of proteins and the effects of nonaqueous solvents on enzyme kinetics. Such understanding will be sought from studies of the model enzyme horseradish peroxidase (HRP). The temporal sequence of changes in secondary and tertiary structure during unfolding will be followed, changes in secondary and tertiary structure and enzyme activity induced by nonaqueous solvents will be correlated, and the dynamics for intraprotein diffusion of substrate analogues will be studied. Experimental methods will include fluorescence and phosphorescence, circular dichroism (CD), and UV/visible absorbance. Eventually, stopped-flow equipment will be sought to permit measurements of faster molecular events. The specific aims are to test five hypotheses. 1) The temporal sequence of changes in secondary structure and tertiary structure during the unfolding of HRP can be determined by monitoring the relative time-course of the tryptophan fluorescence, near/UV CD, far/UV CD, and Soret CD absorbance. 2) Conformational changes in HRP in nonaqueous solutions can be measured and related to the significant reduction of enzyme activity. 3) Trifluoroethanol (TFE) alters the heme accessibility of HRP and alters its enzyme activity. 4) TFE alters the enzyme activity of HRP with other substrates. 5) The structure of substrates and substrate analogues for HRP can be correlated with the activation energy for their diffusion through the substrate channel of HRP.